Process for the preparation of pharmaceutically acceptable salts of (SS,RS) -s-adenosyl-l-methionine

ABSTRACT

The present invention relates to a process for the preparation of pharmaceutically acceptable salts of (SS,RS)-S-adenosyl-L-methionine and allows to obtain the salified (RS)-(+)-S-adenosyl-L-methionine diastereoisomer in amounts lower than or equal to 3% with respect to the salified (SS)-(+)-S-adenosyl-L-methionine diastereoisomer; the salts that can be obtained by the process of the invention keep their configuration stable in time.

[0001] The present invention relates to a process for the preparation ofpharmaceutically acceptable salts of (SS,RS)-S-adenosyl-L-methionine(hereinafter referred to as (SS,RS)-SAMe).

[0002] In particular, the invention relates to a process for thepreparation of pharmaceutically acceptable salts of (SS,RS)-SAMe,wherein the salified (RS)-(+)-S-adenosyl-L-methionine diastereoisomer(hereinafter referred to as (RS)-(+)-SAMe) is produced in amounts lowerthan or equal to 3% with respect to the salified(SS)-(+)-S-adenosyl-L-methionine diastereoisomer (hereinafter referredto as (SS)-(+)-SAMe).

[0003] As it is known, (SS,RS)-SAMe is a physiological methyl donorinvolved in enzymatic transmethylation reactions, that is present in allliving organisms and has therapeutical effects on chronic hepaticdiseases, adiposis, lipaemia, atherosclerosis and it is desirable,therefore, to produce it in high amounts.

[0004] It is also known, (J. W. Cornforth, J.A.C.S., 1977, 99,7292-7300; Stolowitz et al., J.A.C.S., 1981, 103, 6015-6019) that theproducts containing (SS,RS)-SAMe consist of a mixture of twodiastereoisomers: (RS)-(+)-SAMe and (SS)-(+)-SAMe, having the followingstructural formulae:

[0005] Moreover, it was demonstrated (De La Haba et al., J.A.C.S., 1959,81, 3975-3980) that only one of the two diastereoisomers, i.e.(SS)-(+)-SAMe, is enzymatically active for the transmethylation andspontaneously racemises, thereby giving rise to the formation of theinactive diastereoisomer (RS)-(+)-SAMe in a percentage equal to about20% (Wu et al., Biochemistry 1983, 22, 2828-2832).

[0006] The Applicant, in fact, has noted that in all the commerciallyavailable products based on (SS,RS)-SAMe, the inactive diastereoisomer(RS)-(+)-SAMe is present in percentages equal to at least 20%; it wasalso noted that said percentages increase in time even up to 40% andmore.

[0007] This observation clearly confirms that the diasteroisomer mixtureis unstable in time, which, on the other side, had already been noted inrelation with the product in solution (G. L. Creason et al.,Phytochemistry, vol. 24, N. 6, 1151-1155, 1985; H. C. Uzar, Liebigs Ann.Chem. 1989, 607-610).

[0008] The demand for (SS,RS)-SAMe derivatives wherein the percentage ofthe active (SS)-(+)-SAMe diastereoisomer is clearly higher with respectto the inactive (RS)-(+)-SAMe isomer and wherein said percentage turnsout to be stable in time, is particularly felt in the field.

[0009] It was also found that there is an obstacle to the use of(SS,RS)-SAMe and the pharmaceutically acceptable salts thereof at theindustrial level because of their thermal instability, even at roomtemperature, and of the complexity of the preparation and purificationprocesses thereof.

[0010] Several processes for the purification of (SS,RS)-SAMe and forthe production of the pharmaceutically acceptable salts thereof areknown.

[0011] However, the known purification processes, besides providing theuse of strong acid resins (JP 13680/1971) or chelate-type resins (JP20998/1978) or particular and expensive reactants, such as picric orpicolinic acid (U.S. Pat. No. 3,707,536 and U.S. Pat. No. 3,954,726),bring anyhow to the partial racemisation of the sulphur chiral center of(SS,RS)-SAMe and, therefore, lead to final products containing theinactive diastereoisomer in amounts higher than 20%.

[0012] Purification processes that use weak acid resins are also known(JP 14299/1981, FR-A-2531714, EP-A-0141914), which allow, however, toobtain just a partial separation of (SS,RS)-SAMe and, therefore, aninsufficient purity degree for pharmaceutical purposes.

[0013] Even if the realization of some of the above-identified processesenables to obtain a higher purity, the partial racemization implies, atany event, that at least 20% of the inactive diastereoisomer should bepresent; in some cases moreover (FR-2531714), in order to extract theproduct from the cells, there is provided the use of potassiumbicarbonate, with subsequent precipitation of potassium perchlorate,which brings about problems firstly in the separation and then in thedisposal of the product. In EP-A-0141914, the lysis of the cells of theyeast containing (SS,RS)-SAMe is carried out in the presence of anorganic solvent (for example, ethyl acetate, acetone, etc.) by using,moreover, chromatographic columns based on 100-200 mesh resins, withhigh investment and maintaining costs. The use of solvents for theextraction of (SS,RS)-SAMe necessarily implies the employment ofantideflagrant plants and a recovery, distillation and solvent recoverysystems, besides the necessary drying of the exhausted mycelium, inorder to avoid that it is discharged with the residual solvent, allthese factors clearly bringing about additional investment and operationcosts.

[0014] According to a first aspect, the present invention relates to aprocess for the preparation of pharmaceutically acceptable salts of(SS,RS)-SAMe, wherein the salified (RS)-(+)-SAMe diastereoisomer ispresent in amounts lower than or equal to 3% with respect to thesalified (SS)-(+)-SAMe diastereoisomer, which, at a temperature higherthan or equal to 0-12° C., comprises:

[0015] the purification of (SS,RS)-S-adenosyl-L-methionine from enrichedyeast, which shall contain at least 6 g/l thereof, which comprises:

[0016] (a) the adjustment of the pH value to 1.2-3.5;

[0017] (b) the preparation of an aqueous lysate of (SS,RS)-SAMe from theenriched yeast;

[0018] (c) the microfiltration of the resulting lysate;

[0019] (d) the absorption of the resulting microfiltrate on a weak acidresin, by eluting with a 0.1-2 N inorganic acid solution;

[0020] (e) the decolouration of the resulting eluate;

[0021] the concentration of the decolourised eluate, by reverse osmosis,from 30 to 70% by volume;

[0022] the addition of stoichiometric amounts of at least onepharmaceutically acceptable acid salt to the concentrated eluate, so asto obtain the corresponding pharmaceutically acceptable salt of(SS,RS)-SAMe.

[0023] According to a preferred aspect, the so obtained pharmaceuticallyacceptable salt of (SS,RS)-SAMe can be subjected to lyophilization.

[0024] According to another preferred aspect, the process of theinvention is carried out at a temperature of 2-5° C.

[0025] According to a further preferred aspect, the pH value in step (a)is 1-2, whereas the preparation of the lysate in step (b) can take placeby passing the yeast through a breaking-cells equipment, then proceedingwith the microfiltration of the so obtained yeast, for example on aceramic membrane.

[0026] The enriched yeast on which (SS,RS)-SAMe is purified preferablycontains at least 8-10 g/l of (SS,RS)-SAMe; the pharmaceuticallyacceptable acid is selected, preferably, from sulphuric acid andparatoluensulphonic acid.

[0027] It can be noted that the process of the invention allows to useresin/product ratios equal to, for example, 10-20 liters of resin pro kgof absorbed product, which are advantageous with respect to what hasbeen disclosed in JP 20998/1978.

[0028] The process of the invention allows to produce salts of(SS,RS)-SAMe wherein, even at room temperature, it is possible to detecta percentage of the (SS)-(+)-SAMe diastereoisomer equal to at least 97with respect to the (RS)-(+)-SAMe diastereoisomer which is present,accordingly, in percentages lower than or equal to 3.

[0029] The process of the invention allows moreover to exclude the useof organic solvents in the preparation of the lysate, with remarkableadvantages with respect to the purification steps of thepharmaceutically acceptable salts of (SS,RS)-SAMe, as well as ecologicaland environmental advantages.

[0030] It is furthermore possible to obtain a higher yield and purity ofthe pharmaceutically acceptable salts of (SS,RS)-SAMe with respect tothose obtainable by known processes; a purity equal to at least 98% in(SS,RS)-SAMe and a yield equal to at least 90 are obtained, in fact,with respect to the fermented product.

[0031] Thanks to its particular conditions, the process of the inventionallows to avoid the degradation of (SS,RS)-SAMe during the preparationof the lysate and allows to obtain a lysis with a yield higher than 98%and with a content of by-products, the main product of which being5-deacyl-5-methylthioadenosine, lower than 1%.

[0032] (SS,RS)-SAMe, suitably salified as above described, can beproduced, for example, by fermenting a suitable microorganism, such asSaccharomyces pastorianus (ex Saccharomyces carlsbergensis CBS 1513),Saccharomyces cerevisiae (IFO 2044), Torulopsis utilis and Candidautilis.

[0033] The yeast containing (SS,RS)-SAMe can be enriched by theprocesses known in the field, such as for example, the Schlenk methoddescribed in “Journal of Biological Chemistry”, vol. 29, page 1037,(1987), which was modified only in optimizing the use of DL methionineand which was conducted at a maximum temperature of 27,5° C. for about20 hours.

[0034] The (SS,RS)-SAMe-enriched yeast (which, in order to beadvantageously employed in the realization of the present invention,indicatively contains at least 6 g/l of (SS,RS)-SAMe, undergoes, uponadjustment of the pH value to 1.2-3.5, a cellular lysis process, bypassing the yeast, preferably, through a cell-breaking equipment.

[0035] The resulting lysate, after being subjected to microfiltration,for example on a ceramic membrane such as Kerasep® K09A, is adsorbed ona weak acid carboxylic resin, preferably of the cationic type, such asRohm and Haas® IRC86, preferably until saturation (about 150 g/l), andeluted with a solution of an inorganic acid such as, for example, 0.1-2N sulphuric acid, hydrochloric acid, etc.

[0036] The decolouration of the resulting eluate takes then place, forexample by means of a copolymer resin with a styrene-divinylbenzeneunit, such as Resindion® 825L.

[0037] The resulting eluate containing (SS,RS)-SAMe is concentrated, byreverse osmosis, from 30 to 70%, preferably from 40 to 50% by volume.The so obtained concentrate is added with stoichiometrically equivalentamounts of an acid or a mixture of pharmaceutically acceptable acids,such as those indicated above. The so obtained products can be used forpossible preparations in solution or can be subjected to lyophilisation,when one wishes to use them in the solid form.

[0038] The following examples illustrate the invention without limitingit.

EXAMPLE 1

[0039] 1000 kg of yeast obtained by fermentation of Saccharomycescarlsbergensis were enriched with (SS,RS)-SAMe according to the Schlenkmethod, modified as follows. The yeast was added with 100 kg of yeastcream (which, upon dilution with 100 1 of deionised water has a 2.2 g/ltiter), 2 kg of DL methionine, 12 kg of hydrated glucose and 1.5 kg ofcitric acid, keeping under stirring at 27° C. ±0,5° C. for 22 hours,aerating through emission of sterile filtered air at a flow of 0.6l/l/m, thereby obtaining 9 g/l of (SS,RS)-SAMe. After adjusting pH at1.2 by means of H₂SO₄, lysis was carried out, at a temperature of 12°C., by the “Constant Cell Disruption System” produced by Constant SystemLtd., a pressure-type cell-breaking system with a cooling system. Thesolution was then cooled by using first cold water and then brine, untilthe solution was brought to a temperature of about 2° C.

[0040] The obtained mixture was then conveyed to a microfitration plant,endowed with cartridges of the type Verind A-10 HFM 180 SM, forseparating the exhausted solid from the enriched liquid. The panel waswashed with 2000 l of demineralised water at 2° C. The filtration yieldwas 98%.

[0041] The enriched solution was passed through the IRC 86 resin (Rohmand Haas®), a carboxylic resin and eluted with 1 N sulphuric acid, stillkeeping the temperature at about 2° C.

[0042] The collected eluate was decoulorised by using a Resindion® 825Lresin. The enriched solution was concentrated by reverse osmosis until a40% concentration of (SS,RS)-SAMe was obtained. Correspondingstoichiometric amounts of sulphuric acid and paratoluensulphonic acidwere then added to give the disulphate paratoluensulphonate of(SS,RS)-SAMe. The final yield of (SS,RS)-SAMe disulphateparatoluensulphate was 90%.

[0043] The content of (RS)-(+)-SAMe disulphate paratoluensulphonate inthe diastereoisomer mixture of (SS,RS)-SAMe disulphateparatoluensulphonate, analyzed by HPLC, turned out to be 1%. Therelevant data are reported in the following, in the table concerningsample N. 4.

EXAMPLE 2

[0044] 1000 kg of yeast, obtained by fermentation of Saccharomycescarlsbergensis enriched with (SS,RS)-SAMe according to the methoddescribed in EXAMPLE 1, with an activity equal to 8.2 g/kg, were lysatedby a cell-breaking system at a temperature of 12° C. and at a 2 pH.After adding 500 l of water to the resulting solution, themicrofiltration and the subsequent steps were carried out, analogouslyto what described in EXAMPLE 1, washing with 2000 l of colddemineralized water (about 5° C.). 7,5 kA of (SS,RS)-SAMe were obtainedwhich, after being concentrated by reverse osmosis, were salifiedobtaining a 91,4% yield of (SS,RS)-SAMe disulphate paratoluensulphonate(lysis yield: 99%; purification yield: 98%). The time elapsing from theend of the fermentation to the concentration by reverse osmosis was 32hours. The relevant data are reported in the following, in the tableconcerning sample N. 5.

EXAMPLE 3

[0045] The solution obtained by the process of EXAMPLE 1, afterabsorption on IRC 86 (Rohm and Haas®) resin, was eluted with 1 Nsulphuric acid.

[0046] The obtained solution was concentrated up to 20% and then addedwith sulphuric acid and paratoluensulphonic acid in a stoichiometricamount, thereafter it was further concentrated until a 40% solution wasobtained. 14.09 kg RS)-SAMe disulphate paratoluensulphonate were with atransformation yield of 97.8%. The data are reported in the following,in the table to sample N. 6.

EXAMPLE 4 (comparative)

[0047] 3 Samples of SAMIR® [(SS,RS)-SAMe], produced by KnollFarmaceutici S.p.A., were analyzed by HPLC. The values measured for eachsample are as follows:

[0048] SAMPLE 1—100 mg of SAMIR® (vials) batch 045-021; expiration date06/2000. peak retention peak peak area height No. time area height (%)(%) 1 3.661 0.26322 0.00171 0.286 0.410 2 4.246 0.33608 0.00210 0.3650.503 3 4.591 1.82467 0.00906 1.984 2.166 4 5.429 1.00324 0.00573 1.0901.370 5 5.888 51.25485 0.25301 55.715 60.500 6 6.206 37.31255 0.1465840.560 35.051

[0049] SAMPLE 2—200 mg of SAMIR® (tablets); batch 121; expiration date05/2002. peak retention peak area height No. time peak area height (%)(%) 1 3.655 0.35979 0.00221 0.194 0.269 2 4.238 0.40764 0.00265 0.2200.322 3 4.538 3.58281 0.01624 1.932 1.973 4 5.411 1.60136 0.00919 0.8631.116 5 5.828 108.11943 0.52553 58.299 63.833 6 6.144 71.38583 0.2674638.492 32.487

[0050] SAMPLE 3—400 mg of SAMIR® (tablets); batch 040; expiration date10/2002. Peak retention peak area height No. time peak area height (%)(%) 1 3.386 0.03675 0.00041 0.010 0.027 2 5.419 1.40489 0.00853 0.3870.559 3 5.785 214.15843 0.99534 58.973 65.233 4 6.092 147.47305 0.5212540.610 34.162 5 13.468 0.07286 0.00029 0.020 0.019

EXAMPLE 5

[0051] The products obtained according to the process of the inventionin EXAMPLES 1-3, samples 4-6 respectively, were analyzed, similarly towhat has been described in example 4, after four months from the date oftheir production.

[0052] The measured values for each sample were as follows:

[0053] SAMPLE 4 (EXAMPLE 1); batch 003/R. peak retention peak areaheight No. time peak area height (%) (%) 1 2.595 10.89547 0.06085 4.3404.566 2 2.735 7.93823 0.07825 3.163 5.873 3 2.834 8.13165 0.08741 3.2396.561 4 2.946 20.91077 0.12978 8.331 9.740 5 3.355 5.91998 0.02933 2.3582.201 6 3.651 1.91541 0.00909 0.763 0.683 7 4.136 192.60315 0.9289376.728 69.716 8 4.958 1.81995 0.00603 0.725 0.453 9 6.423 0.885890.00276 0.353 0.207

[0054] SAMPLE 5 (Example 2); KF=2.3%; titre=102.6%; batch 001/R. peakretention peak area height No. time peak area height (%) (%) 1 2.5880.23402 0.00223 0.075 0.155 2 2.817 0.08934 0.00099 0.029 0.068 3 2.9080.28759 0.00228 0.092 0.158 4 3.082 6.84701 0.04649 2.194 3.227 5 3.3880.69924 0.00391 0.224 0.272 6 3.697 0.84270 0.00466 0.270 0.323 7 4.224295.93649 1.35851 94.844 94.306 8 5.153 5.50257 0.01712 1.763 1.188 96.696 1.58749 0.00436 0.509 0.303

[0055] SAMPLE 6 (EXAMPLE 3); KF=1.39%; titre=102.7; batch 004/R. peakretention peak area height No. time peak area height (%) (%) 1 2.5840.19250 0.00169 0.058 0.109 2 2.825 0.13395 0.00135 0.041 0.088 3 2.8940.22074 0.00196 0.067 0.127 4 3.060 6.91884 0.04741 2.094 3.072 5 3.3550.82868 0.00484 0.251 0.314 6 3.661 1.53681 0.00907 0.465 0.588 7 4.162313.00031 1.45354 94.736 94.209 8 5.026 5.89462 0.01854 1.784 1.201 96.528 1.66553 0.00450 0.504 0.292

1. A process for the preparation of pharmaceutically acceptable salts of(SS,RS)-SAMe, wherein the salified (RS)-(+)-SAMe diastereoisomer ispresent in amounts lower than or equal to 3% with respect to thesalified (SS)-(+)-SAMe diastereoisomer, which, at a temperature of 0-12°C., comprises: the purification of (SS,RS)-S-adenosyl-L-methionine fromenriched yeast, which shall contain at least 6 g/l thereof, whichcomprises: (a) the adjustment of the pH value to 1.2-3.5; (b) thepreparation of an aqueous lysate of (SS,RS)-SAMe from the enrichedyeast; (c) the microfiltration of the resulting lysate; (d) theabsorption of the resulting microfiltrate on a weak acid resin, byeluting with a 0.1-2 N inorganic acid solution; (e) the decolouration ofthe resulting eluate; the concentration of the decolourised eluate byreverse osmosis, from 30 to 70% by volume; the addition ofstoichiometric amounts of at least a pharmaceutically acceptable acid tothe concentrated eluate, so as to obtain the correspondingpharmaceutically acceptable salt of (SS,RS)-SAMe.
 2. Process accordingto claim 1, wherein the pharmaceutically acceptable salt of (SS,RS)-SAMeis subjected to lyophilization.
 3. Process according to claim 1 or 2,wherein the pH value in step (a) is 1-2.
 4. Process according to any ofthe preceding claims, wherein the preparation of the lysate in step (b)takes place by passing the yeast through a cell-breaking equipment. 5.Process according to any of the preceding claims, wherein thetemperature is 2-5° C.
 6. Process according to any of the precedingclaims, wherein the enriched yeast contains at least 8-10 g/l of(SS,RS)-S-adenosyl-L-methionine.
 7. Process according to any of thepreceding claims, wherein the pharmaceutically acceptable acid isselected from sulphuric acid and paratoluensulphonic acid.
 8. Apharmaceutically acceptable salt of (SS,RS)-S-adenosyl-L-methionineobtainable by the process according to any of the preceding claims.